We are investigating the uses of multiphoton autofluorescence imaging and spectroscopy in tissue for basic biological research and as a potential diagnostic tool for pathologists. Intrinsic tissue fluorescence can potentially provide significant information about the structural and chemical composition of tissue. Multiphoton excitation has an increased potential for excitation of intrinsic fluorophores since emission separation can be very efficient (excitation is far removed from the emission region) and the technique possesses an intrinsic optical sectioning important in scattering tissues. When performing multiphoton microscopy with excitation in the 690-750nm region, tissue autofluorescence becomes prevalent at specimen powers of 10-15mW (~3-fold higher powers than those used for 2PM of conventional fluorophores). The clear advantage of this method is that the tissue is potentially free from any fixation or staining artifacts. Dye penetration through a thick sample is often poor and fixation and dehydration techniques are associated with an ~80% shrinkage, a collapse in which structural relationships are not necessarily preserved. The disadvantage of utilizing autofluorescence is that natural fluorophores are clearly not optimized for their fluorescent properties. Optimization of instrumentation is thus essential for the acquisition of good autofluorescence images.